JP2004533258A - Apparatus and method for pretreating sperm - Google Patents

Abstract

The multi-chamber module (10) for pretreating sperm for assisted reproduction technology comprises a first chamber (40) for holding a fluid medium, a second chamber (52) for containing semen, and a fluid medium for the sperm. A third collection chamber (50) adapted to flow from one chamber (40), from which a fluid medium proceeds into a second chamber (52), whereby sperm Move against the flow of fluid and allow it to move from the second chamber (52) to the third chamber (50) where sperm is collected. A flow limiting barrier comprising a plate with ribs (44) defining a radial passage between the plates is located between the second chamber (52) and the third chamber (50).

Description

[0001]
The present invention thus relates to infertility treatment, and more particularly to IUI (intrauterine insemination), IVF (in vitro fertilization), gamete intratubal transplantation (GIFT), and intracytoplasmic sperm injection (ICSI). Apparatus and method for pretreating sperm for assisted reproductive technologies, including:
[0002]
(Background of the Invention)
To increase the chance of fertilization, several treatments, including IUI, IVF, GIFT and ICSI, are performed with pretreated sperm that consist of viable motile sperm and do not contain seminal plasma and debris. Many methods have been developed to separate motile sperm from semen. The most commonly used sperm separation techniques involving washing and centrifugation (including "swim up" and "Percoll") can cause some damage to sperm (J. Kerlin and W. Byrd "Human" Reprod. "1991; 6: 1241-1246; RJ Aitken and JS Clarkson" J. Reprod. Fertil. "1987; 81: 459-469).
[0003]
More traditional sperm pretreatment methods can cause iatrogenic damage to sperm (D. Mortimer "Human Reprod."1991; 6: 173-176).
[0004]
Aitken and Clarkson have shown that centrifugal force causes the production of reactive oxygen species that can damage sperm and impair fertility. "Percoll" is widely used in laboratory research settings, and its clinical use is associated with certain disadvantages. Certain batches were found to contain high levels of endotoxin. Endotoxins render the batch unsuitable for clinical use (CY Andersen and J. Grinsted, "J. Assisted Reprod. Gent."1997; 14: 624-628). In the latter part of 1996, "Percoll" was withdrawn from clinical use as a sperm separation medium (Guneet Makkar et al., "Fertil. Steril."1999; 72: 796-802).
[0005]
The ideal separation (or isolation) technique is rapid, inexpensive, and separates all motile sperm without damaging them.
[0006]
It has been reported that motile sperm (or motile sperm) aligns rapidly and swims against the flow when sperm is placed in a fluid flow (F. Abed. A new discovery of the phenomenon: Sperm swims against the flow (The new finding of phenomenon in sperm mobility: the spermatozoa swims against flow from In vitro fertilization and reproduction) (Invitro fertilization and reproduction) PCK Leung, Ed., Monduzzi Editor, 1997: pp. 13-15) Non-motile dull sperm are shed downstream along with other cellular components to produce motility. It has been shown that cilia are present in the cells of the endometrium of many mammals; ciliary flow moves in the same direction in both the fallopian tubes and uterus and extends toward the external os of the uterus This flow can be expected to perform two functions: first, it acts as a sperm guide, directing sperm with the correct kinetic parameters to the position of fertilization in an ampule of the fallopian tube. Second, this flow acts as a natural selection mechanism that optimizes the quality of sperm that can reach the position of fertilization.
[0007]
(Summary of the Invention)
It is an object of the present invention to take advantage of the known phenomenon that sperm aligns against flow to enable the sperm to be pre-processed (or prepared) for assisted reproduction techniques and treatment. is there.
[0008]
According to the present invention, there is provided a multi-chamber module for pretreating sperm for assisted reproduction technology, wherein the first chamber holds a fluid medium (or a fluid medium or medium), and contains semen (or Receiving) a second chamber, and a third collection chamber adapted to allow a fluid medium to flow from the first chamber, the third chamber having the fluid medium entering the second chamber from the collection chamber. Wherein a multi-chamber module is provided, wherein the sperm moves against the fluid flow and is moved from the second chamber to the third chamber, from which it is collected as sperm. You.
[0009]
In a preferred embodiment, the second and third chambers are separated by a mechanical flow-restriction barrier through which fluid flows from the third chamber to the second chamber. The sperm moves from the second chamber to the third chamber via the barrier.
[0010]
The barrier may include a stack (or laminate) of spaced plates or disks, defining a passage therebetween.
[0011]
The module also preferably includes control means for controlling the rate of fluid flow from the first chamber to the third chamber.
[0012]
In a preferred embodiment, the second and third chambers are arranged in cup-shaped containers, the chambers being separated by an annular barrier through which the fluid flows.
[0013]
Preferably, the module comprises a waste chamber into which waste can flow from the second chamber, for example via filter means.
[0014]
Further, according to the present invention, there is provided a method of pretreating sperm for assisted reproductive technology, wherein the flow of the fluid medium from the first chamber to the second chamber via the third chamber is performed in the multi-chamber module. Forming, adding a fluid medium to the first chamber, adding semen to the second chamber, and collecting from the third chamber sperm that migrates from the second chamber to the third chamber against the flow of fluid. A method comprising:
[0015]
Preferably, the flow of the broth from the first chamber to the second chamber is controlled such that the flow of the broth in a stable state proceeds from the third chamber to the second chamber.
[0016]
Also, according to the present invention, there is provided a method for forming a fluid flow in a processing module, the method comprising forming a fluid flow, wherein the sperm can move to a collection zone in the module against the flow. A pre-processing method is provided.
[0017]
The device and method of the present invention have several advantages over conventional sperm pretreatment methods. The present invention does not damage sperm because the processing does not require the use of chemicals or centrifuges. The pre-treatment process is quick and simple. The sperm separation process is under direct observation and can be easily controlled. The module can be used by a physician without the need for laboratory equipment. Also, using the module of the present invention not only helps to separate the sperm, but also wash the sperm and therefore does not require any centrifugation process. The present invention can be used not only to separate motile sperm from non-motile sperm, but also to provide sperm suitable for ICSI treatment using morphologically normal sperm having motile ability. Can be done.
[0018]
The present invention will be described in more detail with reference to the accompanying drawings. Throughout the several figures, the same reference numbers refer to the same parts.
[0019]
(Detailed description of preferred embodiment)
Referring to the drawings, the multi-chamber module of the present invention is indicated generally at 10. The module is substantially cylindrical and has an outer peripheral wall 12, a base 14, and a cap 16. It preferably consists of a plastic material. A horizontal dividing wall 18 extends radially inward from the outer peripheral wall 12 and has a radially (or radially) inner surface having an upwardly extending vertical wall 20 contacting the lower surface of the cap 16. As continuous. Immediately below the wall 20 is the outer peripheral wall 22 of a cup-shaped container indicated at 24. This container 24 has a base 26. The upper edge of the outer wall 22 of the container 24 is spaced from the bottom of the vertical wall 20 and defines a circumferential slot therebetween. This slot is closed by a membrane filter 28. Preferably, the filter is such that it allows the passage of only substances smaller than a number in the range of 1.3-3.5 microns, preferably less than 3 microns. The cup-shaped container 24 is supported in the module by, for example, a support member 30 provided on the base 14 of the module. The inside of the support member 30 is connected to the first port 32 and the second port 34 on the outer wall 12 of the module.
[0020]
Two openings 36 and 38 through the base 26 of the container 24 are formed adjacent the center thereof. Opening 36 communicates with outlet port 34 via a valve (not shown). The opening 38 communicates with the outlet port 32 via another valve (not shown). The valve associated with the outlet port 32 is also connected to a chamber 40 defined by walls 12, 18 and 20 and cap 16. This chamber 40 is referred to herein as a culture chamber, that is, a chamber that holds a fluid culture medium.
[0021]
The chamber 42 located below the media chamber 40 and extending below the base of the container 24 is referred to herein as a waste chamber. A vent (not shown) is provided through the upper portion of the outer wall 12 of the waste chamber.
[0022]
Arranged inside the cup-shaped container 24 is a stack of annular plates or disks 44, one of which is shown in detail in FIG. Each of these plates 44 is annular in shape and is spaced from one another by providing a plurality of upstanding ribs 46 on the upper surface of each plate. As shown in FIG. 2, these ribs 46 have a constant thickness, are sector shaped, and extend from the inner diameter wall of each plate 44 toward the outer shape wall. In a preferred embodiment, twenty of these fan-shaped ribs 46 are equally spaced on each plate. When plates 44 are stacked on one another, this provides a radial path across the plate between the ribs, each passage having a depth of, for example, 30-50 microns. In a preferred embodiment, twenty plates 44 are stacked. A cup-shaped container (or receptacle) 48 is provided on the stacked plates 44. The plate 44 rests on the base 26 of the container 24, which is stepped around as seen in FIG. This arrangement of the plate forms a central cylindrical chamber 50, referred to herein as a collection chamber, above the openings 36 and 38, and outside the plate 44, a semen chamber, herein. An outer annular chamber 52 is formed, referred to as
[0023]
As described above, the culture chamber 40 communicates with the collection chamber 50 through the opening 38. The passage between the culture chamber 40 and the collection chamber 50 has a valve associated with (or associated with) the outlet port 32, which is arranged substantially perpendicular to this passage, and is substantially L-shaped. is there. In the horizontal part of the passage, a rod (not shown) made of plastic material with a longitudinally extending groove is arranged on its peripheral surface, along which the fluid medium from the chamber 40 moves to the opening 38. And thus can be moved into the collection chamber 50. The grooves in the rod serve to control the rate of fluid flow from chamber 40 to chamber 50.
[0024]
Next, a method using the module of the present invention will be described. First, a 5 mL syringe is connected to the valve associated with port 32. The syringe is filled with a fluid medium. The valve associated with (or associated with) port 34 is opened and the module is inverted (or inverted). Thereby, about 1.5 mL of the medium is injected into the collection chamber 50 via the opening 38. This continues until there is no air left in the sampling zone. When the collection zone is filled with the broth, the valve associated with port 34 is closed and the module is again inverted, as shown. The syringe is then filled with air and connected to a valve associated with port 32. The module cap 16 is then opened and the media chamber 40 is filled with the fluid media. As a result of the action of gravity, the medium in this chamber 40 flows through the passage linking the chamber with the collection chamber 50, and the fluid is conditioned by a rod having a groove housed in the support member 30. .
[0025]
Then, using a pipette, a small amount of semen is sprayed into the semen chamber 52, ie, outside the stack of plates 44. Then the whole module is placed in the CO ₂ incubator. During the culture period, motile sperm move from the semen chamber 52 to the collection chamber 50 by capillary flow against the flow of fluid, as best shown in FIG. There is already a flow of fluid from the collection zone 50 to the semen chamber 52 between the plates 44 and across the plates 44 because the medium chamber 40 is filled with fluid medium before sperm is added to the semen chamber 52. I do. Due to the nature of motile spermatozoa, they can move from the semen chamber to the collection chamber, while blunt, non-motile sperm cannot withstand fluid flow and do not reach the collection chamber.
[0026]
Semen chamber 52 is gradually filled with fluid. Due to the presence of the membrane filter 28, only seminal plasma and other debris can pass through the filter from the semen chamber 52 and enter the waste chamber 42.
[0027]
After the end of the incubation period, which typically takes about 30 minutes, the cap 16 of the module is completely closed, the valve associated with the opening 34 is opened, and air is injected by a syringe via the port 32 and the associated valve. , And thus into the collection chamber 50. The pressure of the air causes the fluid medium in the collection chamber and the motile sperm therein to exit port 34 and into the sterile tube.
[0028]
Using this module, normal sperm can move against the flow of fluid and pass through the barrier formed by the plate with the ribs. This mechanism acts to select the most qualified sperm and only allows sperm that can move faster than the fluid flow to reach the collection chamber. In this way, motile sperm are separated from seminal plasma, non-motile dull sperm, other cellular components and bacteria.
[0029]
It should be noted that the module of the present invention can also be used to separate morphologically normal sperm from semen, which has motor skills, as required in ICSI procedures.
[Brief description of the drawings]
[0030]
FIG.
FIG. 1 is a partially cutaway sectional view of a preferred embodiment of the collection module of the present invention.
FIG. 2
FIG. 2 is an enlarged view of a part of the guide plate of the module shown in FIG.
FIG. 3
FIG. 3 is an enlarged view of the two lower guide plates of the stack shown in FIG.

Claims (24)

A multi-chamber module for preprocessing sperm for assisted reproduction technology,
A first chamber for holding a fluid medium,
A second chamber containing semen, and a third collection chamber into which the fluid medium flows from the first chamber, from which the fluid medium enters the second chamber. A multi-chamber module comprising: moving spermatozoa against the flow of fluid from the second chamber to a third chamber where they are harvested as sperm. The module of claim 1, further comprising a mechanical flow restriction barrier between the second and third chambers. 3. The module of claim 2, wherein the mechanical barrier means comprises a stack of spaced plates, the stack defining at least one passage between the plates. 4. The module of claim 3, wherein the spaced plates are substantially annular. 5. The module according to claim 3, wherein the plate is provided with spacer ribs. The module according to claim 5, wherein the rib is sector-shaped. 7. The module according to claim 5, wherein each plate is provided with 20 ribs. The module according to any one of claims 3 to 7, wherein the stack comprises 20 plates. 9. The module according to any one of claims 3 to 8, wherein one or each passage has a depth of about 30 to about 50 microns. The module according to any one of claims 1 to 9, further comprising control means for controlling a flow rate of the fluid medium from the first chamber to the third chamber. 11. The module according to claim 10, wherein the control means comprises a rod having a circumferentially extending groove on a peripheral surface thereof, wherein the rod is disposed in a passage of the broth. 12. The module according to claim 10 or 11, wherein the control means is capable of controlling the flow of the broth, resulting in a steady state flow of the broth from the third chamber to the second chamber. 13. The module according to any of the preceding claims, wherein the second and third chambers are arranged in a cup-shaped container and are separated by an annular flow restriction barrier. 14. The module according to any one of claims 1 to 13, further comprising a waste chamber into which waste can enter from the second chamber. The module of claim 14, further comprising a filter means disposed between the second chamber and the waste chamber. 16. The module of claim 15, wherein the filter means allows only materials smaller than 1.3-3.5 microns to pass. 17. The module according to claim 15 or 16, wherein the filter means only allows the passage of materials smaller than 3 microns. The module according to any of the preceding claims, wherein the module is substantially cylindrical. 19. Module according to any of the preceding claims, consisting of a plastic material. 19. A module according to any one of the preceding claims, consisting of glass. A method of collecting sperm,
Providing a multi-chamber module having a first chamber holding a fluid medium, a second chamber containing semen, and a third chamber for collecting sperm;
Supplying a fluid medium to the first chamber;
Forming a flow of the fluid medium from the first chamber to the second chamber via the third chamber;
Supplying the semen to the second chamber;
Culturing the multi-chamber module; and collecting sperm from the third chamber. A method of pre-treating sperm for assisted reproductive technology, comprising forming a flow of a fluid medium from a first chamber to a second chamber via a third chamber in a multi-chamber module, comprising: Adding to the first chamber, adding semen to the second chamber, and collecting sperm from the third chamber that migrates from the second chamber to the third chamber against fluid flow. 23. The method of claim 22, wherein the flow of the broth from the first chamber to the second chamber is controlled such that the steady state flow of the broth proceeds from the third chamber toward the second chamber. A method of pre-treating a sperm, comprising forming a flow in a processing module of a flow of a fluid against which the sperm can move to a collection zone in the module.

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